大腸癌癌化過程硫化poly-LacNAc醣鏈相關基因表現的抑制

Abstract

Poly-LacNAc醣鏈係由重複的N-acetyllactosamine (Gal-GlcNAc; LacNAc)單元組成，是細胞膜表面的主要醣結構之一。若poly-LacNAc醣鏈上的Gal和GlcNAc的六號碳上的氧 (6-O)發生硫酸化(sulfation)，即生成硫化醣鏈(sulfated poly-LacNAc chain)。而過去的研究證實，β-1,3-N-acetylglucosaminyl- transferase 7 (B3GNT7)、carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 6 (CHST6)、β-1,4-galactosyltransferase 4 (B4GALT4) 與carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 (CHST1)醣轉化酶負責形成硫化醣鏈，且solute carrier family 35 (UDP-N-acetylglucosamine(UDP-GlcNAc) transporter), member A3 (SLC35A3)亦參與影響硫化醣鏈的生合成。 消化道系統癌症癌化的過程，會伴隨癌細胞表面醣結構的改變。Sialyl Lewis a (sLea)與sialyl Lewis x (sLex)建構於poly-LacNAc醣鏈末端，為著名的消化道系統相關癌症的腫瘤相關醣抗原 (tumor-associated carbohydrate antigen)。過去的研究證實。sLea和sLex會大量表現於胃癌、胰臟癌及大腸直腸癌的腫瘤組織中，而且具高度轉移能力的大腸癌細胞株的表現量比低轉移能力的細胞株的表現量高。 本實驗室近期的研究發現，B3GNT7在大腸癌腫瘤組織中，經由DNA甲基化，使其表現相較於正常組織的表現有明顯的下降。若使大腸癌細胞株大量表現B3GNT7，不只會降低其細胞表面的sLea醣抗原，同時癌細胞轉移能力也降低許多。因此，我們進一步分析另四個和硫化醣鏈生合成相關的基因，CHST1、CHST6、B4GALT4及SLC35A3在大腸癌腫瘤組織的表現量。我們發現B4GALT4及SLC35A3有較對照組明顯地抑制。此外，我們將HT-29大量表現B4GALT4，其sLea和sLex皆有下降的現象；但是否會因此而抑制癌細胞惡性轉移的特性還需進一步的動物實驗證明。 為了瞭解在大腸癌細胞是經由何種epigenetic機制調控B4GALT4與SLC35A3基因的表現，我們透過NCBI得知這兩個基因的5’端promoter區皆有CpG island，利用亞硫酸鹽定序 (bisulfite sequencing)方法探究是否有甲基化現象，實驗結果顯示，在大腸癌癌化過程可能並不是藉由DNA甲基化抑制B4GALT4與SLC35A3基因。但有趣的是，在大腸癌細胞株經由去甲基化藥物5-aza-2’-deoxycytidine處理後，B4GALT4和SLC35A3表現量有明顯提升，此現象說明此兩基因可能是經由間接的epigenetic機制調控而被抑制。我們進而測試HDAC inhibitor類的epigenetic藥物，觀察此類藥物對於B4GALT4與SLC35A3基因，以及sLea和 sLex醣抗原表現的影響，目的於探索哪些epigenetic藥物能刺激相關硫化醣鏈基因表現，進而抑制大腸癌轉移的可能。

Poly-LacNAc chain, composed of repeating disaccharide unit of N-acetyllactosamine (LacNAc), is one of the major glyco-structures on the cell surface. The sixth carbon positions (C-6) of the Gal and GlcNAc residues of the poly-LacNAc chains can be sulfated, through the activities of carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 (CHST1) and carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 6 (CHST6), respectively, and this transforms the chains into sulfated poly-LacNAc chains. It has been shown that β-1,3-N-acetylglucosaminyltransferase 7 (B3GNT7) and β-1,4-galactosyltransferase 4 (B4GALT4) are responsible for the elongation of sulfated poly-LacNAc chains. In addition, the UDP-GlcNAc transporter, solute carrier family 35, member A3 (SLC35A3) has been shown to be involved in the biosynthesis of sulfated poly-LacNAc chains. The sialyl Lewis a (sLea) and sialyl Lewis x (sLex) glyco-antigens are constructed on the terminals of poly-LacNAc chains. They have been show to play a critical role during carcinogenesis of gastrointestinal cancers, and are well-known tumor-associated carbohydrate antigens. Our recent investigation demonstrated that B3GNT7 gene was significantly down-regulated, through promoter DNA methylation, in colon tumor tissue. Ectopic expression of B3GNT7 gene in colon cancer cell line suppressed the expression of sLea antigen and reduced the metastasis capability of the cells. Based on these previous findings, we aim to further analyze the expression profile of the other four sulfated poly-LacNAc chain-related genes, CHST1, CHST6, B4GALT4 and SLC35A3, during colon cancer oncogenesis. The result showed that the expression of CHST1 and CHST6 genes did not show a consistent up- or down-regulation pattern in colon tumor tissues, while the expression of B4GALT4 and SLC35A3 genes are significantly down-regulated in tumor tissues. Ectopic expression of the B4GALT4 gene in HT-29 colon cancer cells leads to a reduction in the expression of sLea and sLex antigens on cell surfaces. However, the results obtained from bisulfite sequencing showed that the suppression of the B4GALT4 and SLC35A3 genes in colon tumors were not due to promoter DNA methylation. To explore the epigenetic mechanism leading to the suppression of these genes, we treated colon cancer cells with various epigenetic drugs. Interestingly, the data show that DNA methylation inhibitor 5-aza-2’-deoxycytidine markedly induced the expression of both genes in colon cancer cells, suggesting that unidentified mechanism are directly or indirectly involved in the regulation of B4GALT4 and SLC35A3 genes during colon cancer oncogenesis. In future investigations, we plan to investigate the mechanism involved in the suppression of B4GALT4 and SLC35A3 and the functional roles of the expression of sulfated poly-LacNAc chains in the suppression of sLea and sLex antigens in colon cancer.